Milk lacking beta-casein A1

ABSTRACT

A milk which is free of β-casein A 1  protein in the prevention or treatment of coronary heart disease is disclosed. In addition, a process for the testing of DNA from cells obtained from lactating bovines for the presence of DNA encoding certain β-casein proteins, selecting the bovines on the basis of the testing, and then milking those bovines to produce milk free of β-casein A 1  for use in the prevention or treatment of coronary heart disease is disclosed.

FIELD OF THE INVENTION

[0001] This invention relates to the use of milk which is free of theβ-casein A¹ protein in the prevention or treatment of coronary heartdisease. The invention also relates to the testing of DNA from cellsobtained from lactating bovines for the presence of DNA encoding certainβ-casein proteins, selecting the bovines on the basis of the testing,and then milking those bovines to produce milk free of β-casein A¹ foruse in the prevention or treatment of coronary heart disease.

BACKGROUND OF THE INVENTION

[0002] Coronary heart disease is a major cause of death, particularly incountries where the populations are well-nourished, such as in thewestern world. Many factors are implicated as risk factors for thisdisease including obesity, smoking, genetic predisposition, diet,hypertension, and cholesterol.

[0003] Dairy products, especially milk, are a major contributor to thedietary intake of humans, again particularly in western worldpopulations. Milk contains numerous components of nutritional and healthbenefit. Calcium is one example. However, milk is also a significantsource of dietary fat. It is widely accepted that saturated fats foundin milk are a risk factor for coronary heart disease. However, theinventor has discovered an additional risk factor present in some bovinemilk unrelated to the fat content. What is entirely surprising is thesource of the risk. The source is not dependent on the fat content ofmilk. Instead, it is a milk protein, β-casein, which is linked tocoronary heart disease.

[0004] A number of variants of milk proteins have been identified.Initially, three variants of β-casein were discovered (Aschaffenburg,1961) and were denoted A, B and C. It was later found that the A variantcould be resolved into A¹, A² and A³ by gel electrophoresis (Petersonet. al. 1966). The β-casein variants now known are A¹, A², A³, B, C, D,E and F, with A¹ and A² being present in milk in the highestproportions. It is anticipated that other variants may be identified inthe future.

[0005] The inventor has determined that it is the milk protein β-caseinA¹ which represents the risk factor in bovine milk that is linked tocoronary heart disease, or at least is the principal risk factor. Thisdetermination on the part of the inventor forms the basis of the presentinvention.

[0006] There is no relationship between the fat content of milk andβ-casein genotype in cows. Therefore, selecting cattle on the basis ofmilk fat content will not identify which bovines produce the novel riskfactor, namely the specific β-casein variant, in their milk.

[0007] There is no significant difference in the fat content of milkproduced by cows which are homozygous for the β-casein A¹ allele (i.e.A¹A¹) and cows which are homozygous for the β-casein A² allele (i.e.A²A²). This is apparent from studies reported in the literature.

[0008] Ng-Kwai-Hang has carried out several studies. One study(Ng-Kwai-Hang et. al., 1990) suggested that milk containing β-casein A¹rather than β-casein A² may have a slightly higher fat content. However,these differences were very small. The differences between milk from A¹homozygous cows and milk from A² homozygous cows were 0.05% (for thefirst lactation period), 0.07% (for the second lactation period), and0.04% (for the third lactation period).

[0009] In another study, Ng-Kwai-Hang (in an abstract cited by Jakob et.al., 1990) found the opposite effect (i.e. the A¹A¹ product had a lowerfat content than the A²A² product). Thus, the 1995 Ng-Kwai-Hang abstractdirectly contradicts the Ng-Kwai-Hang, et. al., 1990 study.

[0010] McLean et. al., 1984 (McLean) also reported that there was nosignificant difference in the fat content of milk from cows of A¹A¹ andA²A² genotypes (mean±standard error: 45.8±2.6 g/l for milk of A¹A¹ cowsand 48.6±1.9 g/l for milk of A²A² cows).

[0011] Aleandri et. al., 1990 (Aleandri), shows in Table 5 that theleast squares estimates of the effects of different genotypes and theirstandard errors on fat percentage in milk are 0.12±0.09 for A¹A¹ cowsand 0.07±0.09 for A²A² cows. Taking into account the standard error forthe test, Aleandri indicates that the effects of A¹A¹ and A²A² genotypeson milk fat content are equivalent.

[0012] Bovenhuis et. al., 1992 (Bovenhuis), highlights that there arestatistical problems associated with the way in which the genotypeeffects on fat percentages in milk are studied and documented. It isstated that the ordinary least squares estimates may be biased.Bovenhuis points out that the analysis of the effect of a particulargenotype on various characteristics of milk is complex in nature andmay, among other things, be affected by other genes which may be linkedto the gene under study. Bovenhuis attempts to take into account theabove variables and to overcome statistical problems by using an animalmodel method.

[0013] Table 3 of Bovenhuis indicates that, for a statistical model inwhich each milk protein gene is analysed separately and the A¹A¹ cowsdesignated as being the standard (i.e. given a value of 0% fatattributable to the genotype), the A²A² genotype was estimated not tocontribute (i.e. 0%) to the fat content of the milk of the animalsharbouring that genotype when compared to the A¹A¹ genotype. Thestandard error of the test is recorded as 0.02%. Where a statisticalmodel was used in which all milk protein genes were analysedsimultaneously (Table 4 of Bovenhuis) and the A¹A¹ genotype was againdesignated as being the standard (at 0% fat content attributable to theA¹A¹ genotype), the A²A² genotype was estimated to contribute to the fatcontent of the milk at −0.01% when compared with the A¹A¹ genotype. Inthis study a standard error of 0.02 was designated. Taking into accountthe standard error of the tests these results indicate that the A²A²genotype contributes to the fat content of milk in an equivalent mannerto the genotype A¹A¹.

[0014] Gonyon et. al., 1987 reached the same conclusion as Bovenhuis.

[0015] The level of individual components in milk is influenced by boththe genotype and the environment. That is, the variation between animalsin milk output or milk composition is due to both genotypic andphenotypic factors. For example, Bassette et. al., 1988 (Bassette)indicates that the composition of bovine milk may be influenced by anumber of environmental factors and conditions other than geneticfactors. Environmental factors may impact on milk production and theconstituents contained within the milk (including fat content). Forexample, changes in milk composition occur due to:

[0016] the stage of lactation (e.g., the fat content of colostrum isoften higher; the concentration of fat changes over a period of manyweeks as the cow goes through lactation);

[0017] the age of the cow and the number of previous lactations;

[0018] the nutrition of the cow including the type and composition offeed consumed by the cow;

[0019] seasonal variations;

[0020] the environmental temperature at which the cows are held;

[0021] variations due to the milking procedure (e.g., the fat content ofmilk tends to increase during the milking process which means that foran incomplete milking the fat content would generally be lower thannormal and for a complete milking the fat content will be higher thannormal); and

[0022] milking at different times of the day.

[0023] It is therefore apparent from the studies in this field that aperson skilled in the relevant area of technology would not find a linkbetween the fat content of milk and the β-casein genotype of themilk-producing bovines from which that milk is produced.

[0024] Thus, the inventor has for the first time identified the milkprotein β-casein A¹ as a risk factor linked to coronary heart disease inits own right. It is therefore an object of this invention to provide amethod of using milk substantially free of β-casein A¹ to prevent ortreat coronary heart disease or to minimise the risk of developingcoronary heart disease, or to at least provide a useful alternative. Itis also an object of the invention to provide a method of producing milksubstantially free of β-casein A¹ suitable for use in the prevention ortreatment of coronary heart disease or the minimisation of the risk ofdeveloping coronary heart disease, or to at least provide a usefulalternative.

SUMMARY OF THE INVENTION

[0025] In one aspect of the invention there is provided a method ofpreventing or treating coronary heart disease in a human which includesthe step of at least reducing the intake in that human of β-casein A¹.

[0026] Preferably the reduction is effected by the human ingesting milkobtained from one or more lactating bovines, or a product processed fromthat milk, where the milk or product ingested is substantially free ofβ-casein A¹.

[0027] Preferably the milk is substantially free of β-casein A¹ butcontains any one or more of β-caseins A², A³, B, C, D and E.

[0028] More preferably the milk is substantially free of β-caseins A¹, Band C but contains any one or more of β-caseins A², A³, D and E. Mostpreferably the milk is substantially free to of β-caseins A¹, B and Cand contains only β-casein A².

[0029] In a second aspect of the invention there is provided a method ofproducing milk suitable for use in the treatment or prevention ofcoronary heart disease from one or more lactating bovines which milk issubstantially free of β-casein A¹ but which contains any one or more ofβ-caseins A², A³, B, C, D and E, the method including the steps of:

[0030] (i) testing DNA or RNA from cells containing DNA or RNA obtainedfrom the one or more lactating bovines for the presence of DNA or RNAencoding β-casein A¹;

[0031] (ii) selecting bovines which do not have any DNA or RNA encodingβ-casein A¹; and

[0032] (iii) milking the selected bovines.

[0033] In another aspect of the invention there is provided a method ofproducing milk suitable for use in the treatment or prevention ofcoronary heart disease from one or more lactating bovines which milk issubstantially free of β-casein A¹, B and C but which contains any one ormore of β-caseins A², A³, D and E, the method including the steps of:

[0034] (i) testing DNA or RNA from cells containing DNA or RNA obtainedfrom the one or more lactating bovines for the presence of DNA or RNAencoding any one or more of β-caseins A¹, B and C;

[0035] (ii) selecting bovines which do not have any DNA or RNA encodingany one or more of β-caseins A¹, B and C; and

[0036] (iii) milking the selected bovines.

[0037] In another aspect of the invention there is provided a method ofproducing milk suitable for use in the treatment or prevention ofcoronary heart disease from one or more lactating bovines which milk issubstantially free of β-casein A¹ but which contains β-casein A², themethod including the steps of:

[0038] (i) testing DNA or RNA from cells containing DNA or RNA obtainedfrom the one or more lactating bovines for the presence of DNA or RNAencoding β-casein A²;

[0039] (ii) selecting bovines which are homozygous for DNA or RNAencoding β-casein A²; and

[0040] (iii) milking the selected bovines.

[0041] In another aspect of the invention there is provided a method ofproducing milk suitable for use in the treatment or prevention ofcoronary heart disease from one or more lactating bovines which milk issubstantially free of β-casein A¹ but which contains any one or more ofβ-caseins A², A³, D and E, the method including the steps of:

[0042] (i) testing DNA or RNA from cells containing DNA or RNA obtainedfrom the one or more lactating bovines for the presence of DNA or RNAencoding any one or more of β-caseins A², A³, D and E;

[0043] (ii) selecting bovines which have DNA or RNA encoding only forany one or more of β-caseins A², A³, D and E; and

[0044] (iii) milking the selected bovines.

[0045] In another aspect of the invention there is a method of producingmilk suitable for use in the treatment or prevention of coronary heartdisease from one or more lactating bovines which milk is substantiallyfree of β-casein A¹ but which contains β-casein A², the method includingthe steps of:

[0046] (i) testing DNA or RNA from cells containing DNA or RNA obtainedfrom the one or more lactating bovines for the presence of DNA or RNAencoding β-casein A¹ and DNA or RNA encoding β-casein A²;

[0047] (ii) separating bovines which are homozygous for DNA or RNAencoding β-casein A² from bovines which either have DNA or RNA encodingβ-casein A¹ or which have DNA or RNA encoding both β-casein A¹ andβ-casein A²; and

[0048] (iii) milking the bovines which are homozygous for DNA or RNAencoding β-casein A².

[0049] In another aspect of the invention there is provided a method ofproducing milk suitable for use in the treatment or prevention ofcoronary heart disease from one or more lactating bovines which milk issubstantially free of β-caseins A¹, B and C but which contains any oneor more of β-caseins A², A³, D and E, the method including the steps of:

[0050] (i) testing DNA or RNA from cells containing DNA or RNA obtainedfrom the one or more lactating bovines for the presence of DNA or RNAencoding any one or more of β-caseins A¹, B and C and DNA or RNAencoding any one or more of β-caseins A², A³, D and E.

[0051] (ii) separating bovines which have any DNA or RNA encoding anyone or more of β-caseins A¹, B and C from bovines which have DNA or RNAencoding only for any one or more of β-caseins A², A³, D and E; and

[0052] (iii) milking the bovines which have DNA or RNA encoding only forany one or more of β-caseins A², A³, D and E.

[0053] Preferably the one or more lactating bovines of any aspect ofthis invention are Bos taurus bovines.

[0054] More preferably the milk produced according to any aspect of thisinvention is substantially free of β-casein A¹ and the β-caseincontained in the milk comprises greater than 95% by weight β-casein A².

[0055] More preferably the milk produced according to any aspect of thisinvention is substantially free of β-casein A¹ and the β-caseincontained in the milk comprises approximately 100% by weight β-caseinA².

BRIEF DESCRIPTION OF THE DRAWINGS

[0056]FIG. 1 is a graph showing the regression relationship between thedeath rate from Ischaemic Heart Disease (all ages per 100,000 ofpopulation for the year 1985) and the estimated average daily intake ofβ-casein A¹ per head of population (based on country by country dietaryinformation and data on the genotype of the dairy cows in their nationalherds), over a number of countries.

[0057]FIG. 2 is a graph showing the regression relationship between thedeath rate from Ischaemic Heart Disease (per 100,000 males aged 30-69for the year 1985) and the estimated average daily intake of dairyprotein per head of population over a number of countries.

[0058]FIG. 3 is a graph showing the regression relationship between thedeath rate from Ischaemic Heart Disease (per 100,000 males aged 30-69for the year 1985) and the estimated average daily intake of saturatedfat over a number of countries.

[0059]FIG. 4 is a graph showing the regression relationship between thedeath rate from Ischaemic Heart Disease (per 100,000 males aged 30-69for the year 1985) and the estimated average daily intake of red meatover a number of countries.

[0060]FIG. 5 shows the A¹ and A² amplicons for the ACRS genotypingmethod.

[0061]FIG. 6 shows the electrophoresis results for the ACRS genotypingmethod.

[0062]FIG. 7 shows the gene fragment amplified in the Primer Extensiongenotyping method.

[0063]FIG. 8 shows mass spectrometry profile results for the PrimerExtension genotyping method.

DETAILED DESCRIPTION OF THE INVENTION

[0064] This invention is applicable to milk, and all products processedfrom that milk, which milk is substantially free of β-casein A¹.

[0065] As used herein, the term “treatment” in relation to coronaryheart disease means at least a reduction in the risk of a coronary heartdisease event occurring in a human. The terms “treat” and “treating”have equivalent meanings.

[0066] Coronary heart disease means any disease or disorder relating tothe coronary heart system and includes atherosclerosis and ischaemicheart disease.

[0067] The term “Bos taurus” refers to any cow whose pedigree from itsthree prior generations is 50% or more of Bos taurus origin.

[0068] The term “β-casein A¹ allele” is a term used with reference toone of the variant forms of the β-casein gene. Expression of the A¹allele results in the production of the β-casein A¹ protein. Wherereference is made to the presence of the β-casein A¹ allele in anindividual or population, it encompasses both homozygous andheterozygous genotypes with respect to that allele. Similarly, wherereference is made to the presence of β-casein A¹, it encompassesphenotypes resulting from either a homozygous or heterozygous state withrespect to the β-casein A¹ allele.

[0069] An example of an animal which is heterozygous for β-casein is aβ-casein A¹A² bovine. Some animals are homozygous, for example bovinesthat are A¹A¹ for β-casein and those that are A²A² for β-casein. Aβ-casein A²A² bovine is capable of producing only the β-casein A²protein.

[0070] Genetic variation within a species is due at least in part todifferences in the DNA sequence. While there are relatively few suchdifferences in relation to the number of DNA bases or the size of thegenome, they can have a major impact as is evident in the geneticheterogeneity of the human and bovine populations. For example, inbovines, a mutation in the DNA sequence coding for the β-casein proteinat nucleotide position 200 has resulted in the replacement of a cytidinebase with an adenine base. Thus, the triplet codon affected by thischange codes for histidine (CAT) rather than for proline (CCT) at aminoacid position 67 of the protein. Thus, the histidine at position 67results in the cow producing β-casein A¹ while the proline results inthe cow producing β-casein A² (Note: the preceding discussion assumesthat the ancestral bovid expressed β-casein A² and that there are noother DNA variations at other positions on the DNA sequence).

[0071] The term “substantially” as used in the expression “substantiallyfree of β-casein A¹” reflects that it cannot be said with 100% certaintythat a sample of milk is absolutely free of β-casein A¹. On rareoccasions, and despite all efforts to ensure that a herd is β-casein A¹free, an animal capable of producing β-casein A¹ in its milk couldpresent itself in the herd because of a genetic mutation or because ofhuman error. Herds are formed by the genotype testing of animals andthen selecting the desired individuals. All such testing is subject tohuman error. The phrase “substantially free of β-casein A¹” is thereforeintended to account for this. Without the word “substantially”, thephrase would be unduly limiting.

[0072] The products processed from milk that form part of this inventionare derived from a source of bulk milk (i.e. milk from more than oneanimal) and include, but are not limited to:

[0073] (a) bulk milk

[0074] (b) bulk milk used to make cheese whether or not the milk hasbeen pasteurised, sterilised or otherwise treated to reduce the thepopulation of microbes prior to cheese making,

[0075] (c) milk powders,

[0076] (d) milk solids,

[0077] (e) caseins, caseinates, and casein hydrolysates,

[0078] (f) pasteurised, sterilised, preserved milks includingmicrofiitered milks, UHT milks,

[0079] (g) low fat milks,

[0080] (h) modified or enhanced milks,

[0081] (i) ice-cream or other frozen dairy based confections,

[0082] (j) fermented milk products such as yoghurt or quark,

[0083] (k) cheeses including full fat, partial de-fatted and fat-freeprocessed cheeses,

[0084] (I) milk whey,

[0085] (m) food products enriched through the addition of milk productssuch as soups,

[0086] (n) milk from which potentially allergenic molecules have beenremoved,

[0087] (o) confections such as chocolate,

[0088] (p) carbonated milk products, including those with addedphosphate and/or citrate,

[0089] (q) infant formulations which may contain full, partiallyde-fatted or nonfat milk together with a number of additionalsupplements,

[0090] (r) liquid or powdered drink mixtures, and

[0091] (s) buttermilk and buttermilk powder.

[0092] It has been reported that certain human population groups exhibita relatively low incidence of coronary heart disease and certain otherdiseases, notwithstanding the fact that they consume considerablequantities of milk and milk proteins. These people include the Tibetans,rural Gambians, and the Masai and Samburu people of Kenya. The inventorhas identified the fact that a major difference between these populationgroups and other similar population groups is that the milk consumed bythe above people is derived from Bos indicus bovines (e.g. the Zebubreed) and from the Yak (Bos grunniens). Such milk does not containβ-casein A¹.

[0093] In addition, a comparative study in Denmark of the causes ofmorbidity in the Greenland Eskimo population and the predominant Danes,shows very large relative differences that are suggestive of differencesin life-style risk factors. One notable difference is that the Danes arelarge consumers of dairy products whereas the Eskimos are not. Thedifferences in morbidity are illustrated in Table 1 below. TABLE 1Age-adjusted differences in morbidity from chronic diseases betweenGreenland Eskimos and Danes Eskimos/Danes Acute myocardial infarction1/10 Stroke 2/1  Psoriasis 1/20 Diabetes Rare Bronchial asthma 1/25Malignant disorders 1/1  Thyrotoxicosis rare Multiple sclerosis 0Polyarthritis chronica Low

[0094] A further comparison has been carried out using data from thestates of the former West Germany. In this case, the coronary heartdisease death rates have been found to correlate strongly with therelative regional average consumption of β-casein A¹ (Table 2). In thisinstance, the composition of the individual state dairy herds remainedvirtually constant from the 1950's through to the 1980's.

[0095] The data show a remarkable relationship between the relativeincidence of Ischaemic Heart Disease and the relative averageconsumption of β-casein A¹ across the 8 states. This is in markedcontrast to the relatively poor relationships between the incidence ofIschaemic Heart Disease and the recognised listed dietary risk factors.TABLE 2 A comparison of the relative nutritional risk factors forcoronary heart disease and the incidence of Ischaemic Heart Disease(IHD) in the states of the former Federal Republic of Germany(Schleswig-Holstein = 1.00) Relative intake of dietary componentRelative Saturated β-A¹ incidence Fat Cholesterol Alcohol CarbohydratesEnergy casein of IHD Schleswig 1.00 1.00 1.00 1.00 1.00 1.00 1.00Holstein Niedersachsen 0.97 0.96 1.00 0.98 0.99 0.92 0.88 Nordrhein 0.991.02 0.99 1.00 1.02 0.97 1.00 Westfalen Hessen 0.95 0.96 0.98 0.98 0.980.75 0.74 Rheinland-Pfalz 0.95 0.99 1.00 1.02 1.0 0.87 0.78 Saarland0.94 0.93 0.98 1.01 0.98 0.90 0.88 Baden 0.93 1.02 1.02 1.05 1.03 0.500.72 Wurttenburg Bayern 0.96 0.99 1.22 1.06 1.02 0.50 0.74

[0096] A regression relationship between Ischaemic Heart Disease and fatintake was conducted and was shown to be not significant (ρ<0.0684,r²=0.20). However, the regression between Ischaemic Heart Disease andthe intake of β-casein A¹ was highly significant (ρ<0.0001, r²=0.71).The regression relationships are:

IHD=1.56 (±0.79) Fat Intake+86.7 (±74.9)

IHD=81.7 (±13.5) β-Casein A¹−5.4 (±40.4)

[0097] The multiple regression relationship was then generated. In thiscase, the inclusion of both fat intake and β-casein A¹ intake did notimprove the relationship over that with β-casein A¹ alone. The regresionrelationship is:

IHD=78.3 (±15.6) β-Casein A¹+0.259 (±0.557) Fat−19.2 (±51.0)

[0098] The analyses of the relationships between various dietary factorsand Ischaemic Heart Disease outlined in this document indicate thepotential importance of the β-casein variant as a risk factor for heartdisease. The difference between the two casein variants is only oneamino acid. This suggests that the products of proteolysis of thesevariants may be linked to the identified risk factor. Some indication ofthe number, and the major product fragments into which they are split byproteolytic action of a variety of enzymes, is illustrated for theβ-caseins in Table 3. TABLE 3 The β-casein family of proteins FormerRecommended Nomenclature Nomenclature Source of Fragment β-casein A¹β-CN A¹-5P    — β-casein A² β-CN A²-5P    — β-casein A³ β-CN A³-5P    —β-casein B β-CN B-5P    — β-casein C β-CN C-4P    — β-casein D β-CN D-4P   — β-casein E β-CN E-5P    — γ₁-casein A¹ β-CN A¹-1P (f29-209) β-CNA¹-5P γ₁-casein A² β-CN A²-1P (f29-209) β-CN A²-5P γ₁-casein A³ β-CNA³-1P (f29-209) β-CN A³-5P γ₁-casein B β-CN B-1P (f29-209) β-CN B-5Pγ₂-casein A² β-CN A² (f106-209) β-CN A¹-5P or β-CN A²-5P γ_(2’-casein A)³ β-CN A³ (f106-209) β-CN A³-5P γ_(1’-casein B) β-CN B (f106-209) β-CNB-5P γ_(3’-casein A) β-CN A (f108-209) β-CN A¹-5P, β-CN A²-5P or β-CNA³-5P γ₃-casein B β-CN B (f108-209) β-CN B In addition there are anumber of protease peptone components.

[0099] Bovine milk is an important source of proteins and othernutrients required by humans. A high proportion of the common domesticcattle breeds, such as the Holstein, express the β-casein A¹ allele. Forexample, it is estimated that in the late 1980s more than 70% of theCalifornian dairy herd carried the A¹ allele. As noted previously, theβ-casein A¹ variant is of particular interest and therefore, consideringits contribution to milk consumed by the human population in many partsof the world, the proteolysis products of β-casein A¹ are of particularinterest.

[0100] In the graph shown in FIG. 1, the incidence of Ischaemic HeartDisease is plotted against the estimated average consumption of β-caseinA¹ (and its derived proteolysis products). FIG. 1 shows a very strongcorrelation between the consumption of β-casein A¹ and death rate fromIschaemic Heart Disease. In contrast, the correlation with theconsumption of dairy protein (FIG. 2) is much lower. Neither saturatedfat consumption (FIG. 3) nor the consumption of red meat (FIG. 4) showthe strong correlation which the inventor has identified in relation tothe consumption of β-casein A¹, both between countries and withincountries.

[0101] The single amino acid difference between the two predominantβ-casein variants has highlighted the potential role of a difference inthe proteolysis products from different β-casein variants as potentialrisk factors for coronary heart disease. Therefore, the potential impactof pasteurisation is of interest, as prolonged heating is a factor thatis known to influence proteolysis. In particular, this relates to themore severe forms of heat treatment that were used in the early years ofpasteurisation (e.g. Holder pasteurisation which heats milk to 63° C.and holds it for 20-30 minutes). Hence the impact of the introduction ofHolder pasteurisation on the death rate from coronary heart disease isof interest. The inventor has examined the available data and theresults of the analyses are presented in Table 4.

[0102] The analyses reveal a very marked and sudden increase in thedeath rate from coronary heart disease in the four years after theintroduction of Holder pasteurisation. Such data would suggest thepresence of a novel risk factor associated with pasteurisation. It isthe inventor's contention that this risk factor may be associated with aderivative of beta-casein A¹ (for example, a proteolysis product). TABLE4 Comparison of the death rates due to coronary heart disease before andafter the introduction of Holder Pasteurisation in different parts ofthe UK Angina pectoris (AP1) Cerebral embolism Population Holder intro.mort. per mill. and thrombosis (CET) group year AP1 AP2 AP3 Δ % CET1CEL2 CET3 Δ % U.K. Edinburgy 1923 1925  67  92  37^(a) 1924 174  236 36Glasgow 1924 1924  56  91  62^(a) 1924  77  101 31 Dundee 1924 1925  42 64  52^(a) 1925 162  188 16 Aberdeen 1926 1926  91  135  48^(a) 1927121  227 88 Lanarkshire 1935 1937  188  375  99^(b) 1938 153  193 26(excluding 1947 1948  685 1185  73 1948 298  518 74 Glasglow) 1952 19541185 1523  29 1954 518  680 31 Country of Sutherland 1954 1954  963 1710 78 1954 610  823 35 Country of Bute 1956 1956 1610 2848  78 1956 9551398 46 London Admin. 1925 1925  31  112  261^(c) 1926  90  120 33County Average increase  82 42 Norway Oslo 1922 1922   3  43 1333.3^(d)not available

[0103] It is possible, however, that a specific fragment or fragments ofβ-casein A¹ affect the body's immune system as a result of theirimmunosuppressant properties. By reducing or substantially eliminatingthe presence of β-casein A¹ in the diet of an individual, it is believedthat its immune response may be enhanced, or immunosuppression reduced,thereby improving the general well-being of the individual. It isbelieved that some individuals may be particularly susceptible to thepresence of β-casein A¹, and it may be possible to develop a test forsuch susceptible individuals, and to recommend that they reduce oreliminate the consumption of milk or other dairy products containingβ-casein A¹.

[0104] In humans, low density lipoprotein (LDL) oxidation is consideredto be a primary step in the evolution of artherosclerotic damage(Steinberg et. al., 1989). Analysis of protein oxidation productsisolated from atherosclerotic lesions implicates the tyrosyl radical (areactive nitrogen species) and hypochlorous acid in LDL oxidation(Heinecke et. al., 1999). In addition, it has been found (Torreilles andGuerin, 1995) that peptides from bovine casein hydrolysates couldpromote peroxidase-dependent oxidation of human LDLs. The reaction isindependent of free metal ions but requires casein-derived peptides withtyrosyl end residues. This implies that the tyrosyl ending peptide is adiffusable catalyst that conveys oxidising potential from the activesite of the heme enzyme to LDL lipids. Casomorphin-7 is a potentialsource of a tyrosyl radical. It is produced from β-casein A¹ but notβ-casein A² (Jinsmaa et. al., 1999).

[0105] Recognising that dairy products free from β-casein A¹ aredesirable, it is preferable to ensure that the animal from which theproduct is derived has been tested for the presence of the β-casein A¹allele. Subsequent separation of the bovines into separate herds and/orselective breeding programmes (selecting for β-casein A¹ negativeanimals) can be carried out to eliminate the presence of the β-casein A¹from the herd. It will be recognised that such testing may be carriedout in a number of ways without departing from the scope of the presentinvention.

[0106] Any known method for the genotyping of bovines may be used. Suchmethods can be specific for DNA or RNA encoding either β-casein A¹ orβ-casein A². However, general methods which do not specifically test forDNA or RNA encoding β-casein A¹, but additionally test for DNA or RNAencoding other β-caseins, may also be used to form a herd of bovineswhich do not produce β-casein A¹ or produce only β-casein A² in theirmilk.

[0107] For the avoidance of any doubt, any reference to DNA in themethodology of this invention is intended to include cDNA (which is DNAderived from RNA).

[0108] For example, it is known that β-casein A¹ has histidine atposition 67 of the protein whereas β-casein A² has proline at the sameposition. This is due to the presence of an adenine nucleotide atposition 200 of the β-casein DNA. This produces the triplet codon thatspecifies histidine (CAT) rather than proline (CCT). A test whichidentifies the codon that will specify histidine at position 67 of theβ-casein protein can therefore be used to exclude bovines which produceβ-casein A¹ in their milk.

[0109] Similarly, a test which identifies the codon that will specifyproline at position 67 of the β-casein protein can therefore be used toselect bovines which produce β-casein A² (or β-caseins A³, D or E) intheir milk. While a test for animals that are homozygous for thepresence of CCT (that codes for proline) at codon 67 of an animal'sβ-casein gene does not unequivically show whether or not the animal ishomozygous for the β-casein A² allele, the test can show that an animaldoes not possess any of the alleles for β-casein A¹, B and C. Such atest does not need to be any more specific because culling animalsnegative for the test will mean the elimination of β-casein A¹ producinganimals from the herd.

[0110] It is also known that β-caseins B and C, in addition to β-caseinA¹, have histidine at position 67. Also, β-caseins A³, D and E, inaddition to β-casein A², have proline at position 67. Therefore, a testwhich distinguishes between the codons that specify proline andhistidine at position 67 will also distinguish between β-caseins A¹, Band C on the one hand and β-caseins A², A³ ₁ D and E on the other hand.

[0111] For example, while a test for the presence of CAT (histidine) orabsence of CCT (proline) in one or other or both of an animal's allelesat codon 67 of its β-casein gene does not unequivocally show whether ornot the animal contains the β-casein A¹ allele, the test can show thatan animal may contain one or more of the alleles for β-casein A¹, B andC. Such a test does not need to be any more specific because cullinganimals positive for the test (i.e. absence of the proline codon in atleast one allele) will mean the elimination of β-casein A¹ producinganimals from the herd.

[0112] A DNA or RNA test which gives positive identification for animalshomozygous for CCT (proline) at codon 67 can therefore be used toascertain whether a particular bovine does not possess a β-casein A¹allele, whether homozygous or heterozygous. Thus, bovines which dopossess the CCT (proline) at codon 67 at one or both alleles cantherefore be culled from a herd to give a herd which is free of theβ-casein A¹ allele. Milk obtained from that herd therefore cannotcontain β-casein A¹.

[0113] Where it is known that the genetic makeup of the herd is suchthat the only possible alleles possessed by the individuals are forβ-caseins A¹ and A², the culling from the herd of those bovines positivefor histidine at position 67 gives a herd where each individual ishomozygous for the β-casein A² allele. Such a herd will produce milkpossessing only β-casein A².

[0114] The determination of whether the genotype at codon 67 of theβ-casein gene is CCT (proline) or CAT (histidine) can be made by manydifferent methods that are available and which could be used to assayfor this single nucleotide polymorphism (SNP). The methods include DNAsequencing, SSCP (single stranded conformation polymorphism), allelespecific amplification, and assays designed using proprietarychemistries such as Taqman™ (PE Biosystems), Invader™ (Third WaveTechnologies), SnapShot™ (PE Biosystems), Pyrosequencing™(Pyrosequencing AB), Sniper™ (Pharmacia), and DNA chips (hybridisationor primer extension chips).

[0115] The preferred method should have the ability to function wellwith rapidly extracted impure DNA. High test throughput (>1000 ofsamples per day) at low cost is desirable. Since the preferred objectiveis to identify bovines that are homozygous for the β-casein A² allele,the unequivocal positive identification of animals homozygous for CCT atcodon 67 is preferred, rather than simply the absence of a result in atest for the alternative CAT codon.

[0116] Two examples of practical methods for the large scale genotypingof bovines are:

[0117] A manual ACRS (amplification created restriction site) methodwhich can be conducted easily in any molecular genetics laboratory andrequires no specialist equipment or devices. The method can be easilyscaled up to analyse hundreds of samples per day.

[0118] A highly automated method such as the Sequenom™ primer extensionand mass spectrometry system which is capable of analysing thousands ofsamples per day.

[0119] The aim of the ACRS method is to create an amplicon in which onlyone allele of an SNP will form a restriction site. The restriction siteis created by site directed mutagenesis in the amplification step.

[0120] A Dde1 restriction site can be created when the nucleotides CTare present at nucleotide 200 and 201 (positions 2 and 3 of codon 67) ofthe β-casein gene. This would positively identify the presence of theCCT (proline) codon.

[0121] In Example 1 below, the 3′ section of the Casein Dde2 primer hasa mismatch at its penultimate nucleotide (FIG. 5). This is important asit creates a Dde1 restriction site in the A² amplicon only (shown initalics in FIG. 5). In FIG. 5, codon 67 in each template is in boldlowercase. The template is reversed to present the primer in the usual5′-3′ orientation. The mismatch base is underlined.

[0122] Variations of the test could include modification of the sequenceof the 5′ end of the Casein Dde2 primer or 5′ extension of the CaseinDde2 primer with a nucleotide sequence homologous to the β-caseintemplate or 5′ extension of the Casein Dde2 primer with nucleotideswhich are not homologous to the β-casein sequence. The second primer forthe ACRS is less critical and many compatible primers could be used. Theprimer known as Casein4 5′-CCTTCTTTCCAGGATGAACTCCAGG-3′ (SEQ ID NO: 2)has been found to be the most effective.

[0123] PCR amplification with this pair of primers produces a 121 basepair fragment in all β-casein alleles. However, the definitivediagnostic step is that only alleles with CT at positions 200 and 201(i.e. specifying amino acid 67 of the β-casein) can be cut with therestriction enzyme Dde1. This produces distinctive 86- and 35-base pairfragments.

[0124] The first step of the primer extension method is PCRamplification of the region of the β-casein gene containing codon 67. InExample 2 below, a 319 bp fragment (shown in FIG. 7), was amplified. InFIG. 7, the primer regions are shown underlined. Alternate bases of theSNP are shown bracketed.

[0125] The post PCR product is cleaned with a SAP reaction to removeunincorporated dNTPs. An extension primer complementary to the bolditallicised sequence is added to the cleaned product along with anextension mixture containing ddA, ddC, ddT and dG. The following sizeextension products are obtained: Name Sequence (5′-3′) SEQ ID NO: Mass(Da) Primer AGR-RMA6 GTTTTGTGGGAGGCTGTTA 3 5920.90 Contaminant (Pause)GTTTTGTGGGAGGCTGTTAG 4 6250.10 Analyte A GTTTTGTGGGAGGCTGTTAT 5 6209.10Analyte C GTTTTGTGGGAGGCTGTTAGGGA 6 7205.70

[0126] If codon 67 of β-casein is CAT, a 20 bp, 6209.10 Dalton productis obtained, whereas if the sequence is CCT, a 23 bp, 7205.70 Daltonproduct is obtained. These products can be clearly distinguished andseparated from possible contaminants by MALDI-TOF mass spectrometry.

[0127] The results of the genotype testing obtained from either methodare then used to select bovines positively identified as having CCT(proline) at position 67 at both alleles. Such bovines cannot produceβ-casein A¹ in their milk. The selected bovines are kept in a separateherd and are milked separately. Ideally the milk from that separate herdis kept separate from other milk which may contain β-casein A¹.

[0128] The selected bovines may be uniquely identified (e.g.alternatives include ear-tagging with a unique tag, or use of anelectronic tag or use of a specific tag that identifies the bovine asbeing free of the β-casein A¹ allele or branding for futureidentification). The selected bovines are milked to give milk free ofβ-casein A¹. Preferably, the milk is phenotype tested to confirm thatthe milk is substantially free of β-casein A¹.

[0129] A bulk quantity of milk from the selected bovines may then beprocessed into one or more milk products, such as fresh milk, cheeses,yoghurts, milk powders etc.

[0130] Finally, it will be appreciated that various other alterationsand modifications may be made to the foregoing without departing fromthe spirit or scope of this invention.

EXAMPLES Example 1

[0131] ACRS Method

[0132] At least 10 hairs were pulled from the end of the tail switch ofa cow so that the hook-shaped follicles were retained on the end of theremoved hairs. This was achieved easily by pulling the tail hairs upwardwhile holding the rest of the switch down. If the tail has been docked,longer hairs from the end of the docked tail or other locations on thebody may be substituted. Tail hairs are preferred.

[0133] Five hair follicles from one cow were cut into a sterile 1.5 mlmicrofuge tube. Solution A (200 μl) was added to the tube and the tubeplaced in a boiling water bath for 15 minutes. The tube was removed andSolution B (200 μl) added followed by mixing.

[0134] Solution A (200 mM NaOH)

[0135] Solution B (100 mM Tris-HCl, pH 8.5 with an extra 200 mMHCl)—prepared by combining 1 M Tris-HCl, pH 8.5 (10 ml) with conc. HCl(1.67 ml) and making up to

[0136] 100 ml with distilled water.

[0137] Crude DNA extract (1.5 μl) from hair follicles (prepared asabove) or DNA (20-50 ng) (prepared by another method) was transferred toa well of a 96-well PCR plate. PCR cocktail (20 μl) was added to thewell. The well was overlayed with mineral oil and centrifuged briefly toremove air bubbles.

[0138] The PCR cocktail was prepared according to the following:Components Final Concentration 10X PCR Buffer minus Mg 20 mM Tris-HCl(pH 8.4), (GibcoBRL ®): 50 mM KCl 2 mM dNTPs mixture (GibcoBRL ®): 0.2mM each 50 mM MgCl₂ (GibcoBRL ®): 1.3 mM Primers: 20 μM Casein4 0.5 μM20 μM CaseinDde2 0.5 μM Taq DNA Polymerase 5 U/μl 0.75 units perreaction (GibcoBRL ®):

[0139] The primers used are: (SEQ ID NO: 2) Casein45′-CGTTCTTTCCAGGATGAACTCCAGG-3′ (SEQ ID NO: 1) CaseinDde25′GAGTAAGAGGAGGGATGTTTTGTGGGAGGCTCT-3′

[0140] PCR was carried out on an MJ Research PTC200 (hot bonnet) usingthe following protocol:  1 cycle 94.0° C.  4 mm 35 cycles 94.0° C. 30sec Denature 60.0° C. 30 sec Anneal 72.0° C. 30 sec Extend  1 cycle72.0° C.  4 mm end  4.0° C.

[0141] Following PCR, restriction enzyme cocktail (10 μl) was added andthe mixture incubated at 37° C. overnight. The restriction enzymecocktail was prepared according to the following: Components FinalConcentration Dde I 10 U/μl (GibcoBRL ®) 4.5 units per reaction REACT ®3 (GibcoBRL ®) 25 mM Tris-HCl (pH 8.0), 5.0 mM MgCl₂, 50 mM NaCl

[0142] The amplification product (10 μl) was analysed by electrophoresis(80V, 1 hour) in ethidium bromide stained agarose gel (3%, 1× TBE).

[0143]FIG. 6 shows the results of 20 samples analysed by the procedureoutlined above.

[0144] A size standard ladder was loaded in position 0. The 100 bp bandis identified in FIG. 6. The negative control (no DNA) was loaded inposition 20. Samples homozygous for CT at positions 2 and 3 of codon 67of the β-casein gene result in a single 86 bp band when cut by Dde1.This is shown in load positions 1,2,10,11,12,13,14, and 17. Samples notcontaining CT at positions 2 and 3 of codon 67 of the β-casein gene arenot cut by Dde1, leaving a single 121 bp band. This is shown in loadpositions 4,5,7 and 9.

[0145] Heterozygous samples result in both cut (86 bp) and uncut (121bp) bands. This is shown in load positions 3,6,8,15,16,18 and 19.Because of heteroduplex formation, the uncut band (121 bp) is expectedto be more intense than the cut band (86 bp).

Example 2

[0146] Primer Extension Method

[0147] DNA extracts from hair follicles were prepared using the methoddescribed in Example 1. Alternatively, genomic DNA isolated by othermethods can be used at a concentration at about 2.5 ng/μl.

[0148] A DNA sample (1 μl) from each of 96 animals was placed into a 96well PCR microtitre plate (or alternatively, from each of 384 animalsinto a 384 well PCR plate).

[0149] For the 96 well plate, a cocktail of the following reagents wasprepared in a 1.5 ml microtube. The cocktail (4 μl) was added to eachwell in the plate with a repeating pipette. Reagent Volume μl Water(HPLC grade) 222 10x Hotstar Taq PCR buffer 50 containing 15 mM MgCl₂HotStar Taq Polymerase (5 U/μl) 4 25 mM MgCl₂ 20 dNTP 25 mM 4 Forwardand reverse primer mix 100 Forward: actggattatggactcaaagatttg (SEQ IDNO: 7) Reverse: aaggtgcagattttcaacat (SEQ ID NO: 8) (1 μM each primer)

[0150] PCR was carried out using the following protocol:  1 cycle: 95°C. 15 minutes 45 cycles: 95° C. 20 seconds 56° C. 30 seconds 72° C. 1minute  1 cycle: 72° C. 3 minutes end  4° C.

[0151] The following SAP solution was prepared in a 1.5 ml microtube:Reagent Volume μl Nanopure water 792.54 hME Buffer (Sequenom, San 88.06Deigo) Shrimp alkaline phosphatase 155.4

[0152] The solution was mixed well and centrifuged for ten seconds at5000 RPM.

[0153] SAP solution (2 μl) was transferred to each well of the platecontaining the samples. The plate was incubated using a thermocycler at37° C. for 20 minutes, 85° C. for 5 minutes, and then holding at 4° C.

[0154] The following extension cocktail was prepared in a 1.5 μlmicrotube: Reagent Volume μl Nanopure Water 895.11 μl Sequenom 10x hMEextend buffer with 2.25 103.6 μl mM ddA, ddC, ddT, dG Primer (100 uM)27.97 μl RMA6 R: gttttgtgggaggctgtta (SEQ ID NO: 9) Thermosequenase (32U/μl) 9.32 μl

[0155] The extension cocktail (2 μl) was added to each well of thesample plate. The plate was sealed and thermocycled as follows:  1cycle: 94° C. for 2 minutes 40 cycles: 94° C. for 5 seconds 52° C. for 5seconds 72° C. for 5 seconds End  4° C.

[0156] Prior to mass spectrometry the samples were cleaned usingSpectroCLEAN and then analysed using MALDI-TOF MS.

[0157] The profiles obtained for homozygous and heterozygous animals forthe CCT and CAT SNPs are shown in FIG. 8. The location of the primer,analyte A and analyte C extension products are shown.

[0158] Industrial Application

[0159] The invention provides a useful food product capable ofincreasing the health of an individual, or the health of a population.The invention relates to a method of preventing or treating coronaryheart disease in a human population which derives some of its foodintake from milk or other dairy products by reducing or substantiallyeliminating the presence of β-casein A¹ within the diet of thatpopulation.

[0160] References

[0161] 1. Aleandri, R., Buttazzoni, L. G., Schneider, J. C., Caroli, A.,and Davoli, R. (1990) J. Dairy Sci., 73, 241-255.

[0162] 2. Aschaffenburg, R. (1961) Nature, 192, 431-432.

[0163] 3. Bassette, R., and Acosta, J. S. (1988) Fundamentals of DairyChemistry, 3^(rd) Ed.,—Chapter 1: Composition of Milk (Ed. Wong, N. P.)Van Nostrand Reinhold, New York, pp 1-38.

[0164] 4. Bovenhuis, H., van Arendonk, J. A. M., and Korver, S. (1992)J. Dairy Sci., 75, 2549-2559.

[0165] 5. Gonyon, D. S., Mather, R. E., Hines, H. C., Haenlein, G. F.W., Arave, C. W., and Gaunt, S. N. (1987) J. Dairy Sci., 70, 2585-2598.

[0166] 6. Heinecke, J. W. (1 999) FASEB J., 13, 1113-1120.

[0167] 7. Jakob, E. and Puhan, Z. (1997) Bulletin of the IDF, 304, pp2-3 and 6-8.

[0168] 8. Jinsmaa, Y. and Yoshikawa, M. (1999) Peptides, 20, 957-962

[0169] 9. McLean, D. M., Graham, E. R. B., Ponzoni, R. W., and McKenzie,H. A. (1984) J. Dairy Res., 51, 531-546.

[0170] 10. Ng-Kwai-Hang, K. F., Monardes, H. G., and Hayes, J. E.,(1990) J. Dairy Sci., 73, 3414-3420.

[0171] 11. Peterson, R. F., and Kopfler, F. C. (1966) Biochem. Biophys.Res. Commun., 22, 388-392.

[0172] 12. Steinberg, D., Parthasarathy, S., Carew, T. E., Khoo, J. C.and Witzum, J. L. (1989) N. Engl. J. Med., 320, 915-924.

[0173] 13. Torreilles, J. and Guerin, M. C. (1995) French Compt. RenduSeances Soc. Biol. Filial, 189, 933-945.

[0174]

1 14 1 33 DNA Artificial Sequence Description of Artificial SequencePrimer 1 gagtaagagg agggatgttt tgtgggaggc tct 33 2 25 DNA ArtificialSequence Description of Artificial Sequence Primer 2 ccttctttccaggatgaact ccagg 25 3 19 DNA Artificial Sequence Description ofArtificial Sequence Primer 3 gttttgtggg aggctgtta 19 4 20 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 4gttttgtggg aggctgttag 20 5 20 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 5 gttttgtggg aggctgttat 206 23 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 6 gttttgtggg aggctgttag gga 23 7 25 DNAArtificial Sequence Description of Artificial Sequence Primer 7actggattat ggactcaaag atttg 25 8 20 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 8 aaggtgcaga ttttcaacat 20 9 19 DNAArtificial Sequence Description of Artificial Sequence Primer 9gttttgtggg aggctgtta 19 10 46 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 10 gagtaagagg agggatgttttgtgggaggc tcttagggat gggccc 46 11 46 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 11gagtaagagg agggatgttt tgtgggaggc tcttagtgat gggccc 46 12 318 DNAArtificial Sequence Description of Artificial Sequence Syntheticnucleotide sequence 12 actggattat ggactcaaag atttgttttc cttctttccaggatgaactc cggataaaat 60 ccaccccttt gcccagacac agtctctagt ctatcccttccctgggccca tccmtaacag 120 cctcccacaa aacatccctc ctcttactca aacccctgtggtggtgccgc ctttccttca 180 gcctgaagta atgggatctc caaagtgaag gaggctatggctcctaagca maaagaaatg 240 cccttcccta aatatccagt tgagcccttt actgaaagscagagcctgac tctcactgat 300 gttgaaaatc tgcacctt 318 13 56 DNA Bos sp. 13cccttgggcc catccctaac agcctcccac aaaacatccc tcctcttact caaacc 56 14 57DNA Bos sp. 14 cccttgggcc catccataac agcctcccac aaaacatccc tcctcttactcaaaccc 57

1. A method of preventing or treating coronary heart disease in a humanwhich includes the step of at least reducing the intake in that human ofβ-casein A¹.
 2. A method as claimed in claim 1 wherein the reduction iseffected by the human ingesting milk obtained from one or more lactatingbovines, or a product processed from that milk, where the milk orproduct ingested is substantially free of β-casein A^(l).
 3. A method asclaimed in claim 2 wherein the milk is substantially free of β-casein A¹but contains any one or more of β-caseins A², A³, B, C, D and E.
 4. Amethod as claimed in claim 2 wherein the milk is substantially free ofβ-caseins A¹, B and C but contains any one or more of β-caseins A², A³,D and E.
 5. A method as claimed in claim 2 wherein the one or morelactating bovines are or include Bos taurus bovines.
 6. A method asclaimed in claim 2 wherein the milk produced is substantially free ofβ-casein A¹ and the β-casein contained in the milk comprises greaterthan 95% by weight β-casein A².
 7. A method as claimed in claim 2wherein the milk produced is substantially free of β-casein A¹ and theβ-casein contained in the milk comprises approximately 100% by weightβ-casein A².